Hidalgo-Carcedo, C. | Search bioRxiv

bioRxiv2025

Allosteric and Energetic Remodeling by Protein Domain Extensions

Many functions of proteins are performed by independently folding structural units called domains. The structures of domains are conserved during evolution but they are not identical. For example, the >270 human PDZ domains vary in the number of secondary structure elements and in the length of loops. An important but largely unexplored question is the impact of these extensions on protein energy landscapes: beyond any immediate functional effects, do extensions also alter the consequences of perturbations elsewhere in the domain, altering the potential for regulation and evolvability? Here we perform massively parallel energetic measurements on a model human PDZ domain to directly and comprehensively answer this question. In total we quantify the binding to a ligand and abundance of [~]190,000 protein variants to quantify free energy changes for mutations throughout the canonical domain fold and [~]7,000 energetic couplings between these mutations and the two domain extensions, both alone and in combination. We find that both extensions--one structured and one more dynamic--substantially and specifically re-shape the energy landscape of the domain, with the removal of an [a]-helix altering the energetic consequences of 424 mutations in 54 sites on fold stability and 420 mutations in 56 sites on binding to a ligand. These changes to the energy landscape alter the effects of 330 allosteric mutations, including at solvent-accessible surface sites. Extending or pruning the domain therefore reshapes its energetic and allosteric landscape, adding and removing opportunities for the allosteric control of protein function.

biophysics↗

bioRxiv2021

Global mapping of the energetic and allosteric landscapes of protein binding domains

Allosteric communication between distant sites in proteins is central to nearly all biological regulation but still poorly characterised for most proteins, limiting conceptual understanding, biological engineering and allosteric drug development. Typically only a few allosteric sites are known in model proteins, but theoretical, evolutionary and some experimental studies suggest they may be much more widely distributed. An important reason why allostery remains poorly characterised is the lack of methods to systematically quantify long-range communication in diverse proteins. Here we address this shortcoming by developing a method that uses deep mutational scanning to comprehensively map the allosteric landscapes of protein interaction domains. The key concept of the approach is the use of multidimensional mutagenesis: mutational effects are quantified for multiple molecular phenotypes--here binding and protein abundance--and in multiple genetic backgrounds. This is an efficient experimental design that allows the underlying causal biophysical effects of mutations to be accurately inferred en masse by fitting thermodynamic models using neural networks. We apply the approach to two of the most common human protein interaction domains, an SH3 domain and a PDZ domain, to produce the first global atlases of allosteric mutations for any proteins. Allosteric mutations are widely dispersed with extensive long-range tuning of binding affinity and a large mutational target space of network-altering edgetic variants. Mutations are more likely to be allosteric closer to binding interfaces, at Glycines in secondary structure elements and at particular sites including a chain of residues connecting to an opposite surface in the PDZ domain. This general approach of quantifying mutational effects for multiple molecular phenotypes and in multiple genetic backgrounds should allow the energetic and allosteric landscapes of many proteins to be rapidly and comprehensively mapped.

biophysics↗